Mycobacterium tuberculosis is a major opportunistic pathogen in patients with HIV-AIDS. Current tuberculosis treatment regimes are severely hampered by the occurrence of multidrug resistant strains of M. tuberculosis and there is a critical need for the development of novel chemotherapeutics. In this proposal we initiate studies on the menaquinone biosynthesis pathway in M. tuberculosis. Menaquinone is the sole quinone in the mycobacterial electron transport chain and, since the pathway leading to the biosynthesis of menaquinone is absent in humans, the bacterial enzymes catalyzing the synthesis of menaquinone from chorismate are potential novel targets for drug discovery. The biosynthesis of menaquinone has been extensively studied in E. coli and B. subtilis. Homologs of all but one of the E. coil enzymes are present in M. tuberculosis. In Specific Aim 1 we will clone, express and purify all of the putative menaquinone biosynthetic enzymes from M. tuberculosis. We will then determine if the mycobacterial enzymes catalyze the reactions that have been predicted for them based on sequence homology. The availability of recombinant, active enzymes with known function is essential for the detailed enzymological studies that will form the basis of future efforts to rationally design compounds targeted at menaquinone biosynthesis in M. tuberculosis. In order to determine which menaquinone biosynthetic enzymes will be suitable for future inhibitor design, we need to know whether inhibition of a specific enzyme will impact the viability of the bacterium. In Specific Aim 2 we will use gene replacement methods to knockout individual menaquinone genes and determine the effect of the knockouts on bacterial survival. Knowledge of the phenotype caused by inactivation of a specific enzyme through knockout experiments will be an important reference point for future studies when the effect of specific enzyme inhibitors on the bacterium are evaluated.